1. Field of the Invention
The present invention relates generally to the field of cytokine actions and more particularly concerns methods and compositions for inhibiting and modulating the actions of CXC intercrine molecules. Disclosed are peptide compositions which inhibit interleukin 8 (IL-8) and, particularly, which preferentially inhibit IL-8-induced release of degradative enzymes by neutrophils. These compositions may be employed to treat various inflammatory diseases and disorders including the Adult Respiratory Distress Syndrome (ARDS) and cystic fibrosis.
2. Description of the Related Art
IL-8 is a member of the CXC intercrine family of cytokines, so named due to elements of their N-terminal sequences. This family also includes, amongst others, peptide molecules known as growth related oncogene (GRO, or GRO/MGSA) and macrophage inflammatory protein 2.beta. (MIP2.beta.). IL-8 is a peptide of approximately 8 kD, and is about 72 amino acids in length, with this length varying according to the post-translational processing in different cell types (Yoshimura et al., 1989; Hebert et al., 1990; Strieter et al., 1989). The IL-8 gene was first identified by analyzing the genes transcribed by human blood mononuclear cells stimulated with Staphylococcal enterotoxin A (Schmid and Weissman, 1987). IL-8 production is known to be induced by tumor necrosis factor and interleukin 1 (Strieter et al., 1990).
MGSA/GRO.alpha. is a peptide which was first identified as an autostimulatory growth factor of Hs 294T melanoma cells (Richmond et al., 1983; 1985; Richmond and Thomas, 1986). Further studies indicated that it was produced by diverse melanoma cell lines (Lawson et al., 1987) and played an important role in the tumorigenesis and growth of malignant melanoma cells (Mintz and Silvers, 1993). Sequence analysis demonstrated that MSGA/GRO.alpha. is a member of the superfamily called ".alpha.-chemokines" (Oppenheim et al., 1991). This family of proteins were chemotactic for neutrophils and had substantial sequence homology, a C-X-C motif near the amino-terminal end, and two additional Cys residues closer to the carboxyl-terminal end. The functions of a chemokines were mediated by receptors on the cell surface membrane (Murphy and Tiffany, 1991; Holmes et al., 1991; Mueller et al., 1994). Recent studies showed the presence of some types of chemokine receptors on melanoma cells. Some melanoma cells possess interleukin-8 (IL-8) receptors similar to those on neutrophils (Holmes et al., 1991; Mueller et al., 1994; Moser et al., 1993; Norgauer et al., 1996).
MGSA/GRO.alpha. is a 73 amino acid peptide which shares sequence characteristics of a superfamily of peptides called .alpha.-chemokines. Richmond and colleagues initially discovered that melanoma cells secreted autostimulatory (autocrine) growth factors (Richmond et al., 1983; 1985; Richmond and Thomas, 1986; Richmond et al., 1982). It was found that most of the activity was caused by a single acid stable protein of about 15 kDa and designated it melanoma growth stimulatory activity (MGSA) (Richmond et al., 1983; 1985). MGSA was found to be a mitogen for the melanoma cell line Hs 294T which produces this factor. MGSA was secreted by diverse melanoma cell lines but not by benign nevus cell lines (Lawson et al., 1987), while immunoreactive MGSA was shown in both types of cells (Richmond et al., 1986). cDNA for MGSA isolated from Hs 294T cells was later found to be identical to oncogene growth-related peptide (GRO.alpha.) gene (Richmond et al., 1988). The formal name for this protein was then designated as MGSA/GRO.alpha..
Recently, a second chemokine was found to be important for melanoma cell growth and metastasis in some melanoma cell lines. Schadendorf and colleagues determined that some melanoma cell lines tested secreted IL-8 (Schadendorf et al., 1993). Both of two IL-8 secreting cell lines studied in more detail were dependent on IL-8 for growth. Antisense oligonucleotides targeted against human IL-8 mRNA inhibited cell proliferation, colony formation in soft agar, and secretion of IL-8 into culture supernatants. In an analysis of 13 different human melanoma cell lines, it was shown that expression of IL-8 correlates with the metastatic potential of melanoma cells in BALB/c nude mice (Singh et al., 1994).
Other studies further indicate a role of these chemokines in melanoma growth and tumorigenesis. Mintz and Silvers developed a method of producing melanomas by grafting skin from Tyr-SV40E transgenic mice which are highly susceptible to melanoma to Tyr-SV40E hosts of a low susceptibility of the same inbred strain (Mintz and Silvers, 1993). It was suggested that growth factors and cytokines known to be produced in wound repair may trigger the growth and malignant conversion of melanocytes. Nanney and colleague showed that MGSA/GRO.alpha. and its receptors are present in human bum wounds and may act as a mediator for wound repair (Nanney et al., 1995). MGSA/GRO.alpha. and I11-8 was induced by ultraviolet B radiation in human keratinocyte cell lines (Venner et al., 1995) IL-8 interacts with at least two distinct receptors on neutrophils (Holmes et al., 1991; Murphy and Tiffany, 1991). The receptors are coupled to GTP-binding proteins, allowing transmission of the IL-8 signal into the cell (Wu et al., 1993). While most of the members of the intercrine family, such as GRO and MIP2.beta., bind to one of the receptors, IL-8 binds to both of the IL-8 receptors (LaRosa et al., 1992; Cerretti et al., 1993). The three dimensional structure of IL-8 has been elucidated by NMR (Clore et al., 1990) and by X-ray crystallography (Clore and Gronenbom, 1992; Baldwin et al., 1991). A freely movable amino terminal end is followed by three beta pleated sheets and an alpha helix is located at the carboxyl-terminal end (Oppenheim et al., 1991). Several lines of evidence suggest that both the amino- and carboxyl-terminal ends are involved in binding to its receptors (Clore et al., 1990; Clark-Lewis et al., 1991; Moser et al., 1993).
Certain functions of the CXC intercrines have been elucidated by several laboratories (Yoshimura et al., 1989; Schroder et al., 1988; Peveri et al., 1988). For example, the major functions of the IL-8 peptide appear to be related to its ability to stimulate neutrophil chemotaxis and activation (Larsen et al., 1989; Schroder et al., 1988; Peveri et al., 1988; Yoshimura et al., 1987) and to promote angiogenesis (Koch et al., 1992). If neutrophils are `primed`, e.g., by agents such as surface adherence or E. coli endotoxin (also known as lipopolysaccharide or LPS), IL-8 also stimulates the release of neutrophil enzymes such as elastase and myeloperoxidase.
Although the neutrophil inflammatory response is essential for the destruction of bacteria which are invading the body, inappropriate neutrophil activation causes several problems. For example, if the neutrophils are properly primed when attracted to the lungs, they release destructive enzymes into the lung tissue. This can lead to the development of adult respiratory distress syndrome (ARDS) (Weiland et al., 1986; Idell et al., 1985). ARDS attacks between 150,000 and 200,000 Americans per year, with a mortality rate of 50-80% in the best clinical facilities (Balk and Bone, 1983). ARDS is initiated by bacterial infections, sudden severe dropping of the blood pressure (shock), and many other insults to the body. Recent studies have demonstrated that IL-8 is the major neutrophil activator in the lungs of patients with ARDS (Miller et al., 1992), and primate models of endotoxin shock also implicate IL-8 as a causative agent (Van Zee et al., 1991).
High concentrations of IL-8 have also been found in inflammatory exudates in other disorders and pathological conditions in which IL-8 is thought to play an important pathogenic role (Brennan et al., 1990; Miller and Idell, 1993; Miller et al., 1992). For example, IL-8 has also been implicated as a possible mediator of inflammation in rheumatoid arthritis (Brennan et al., 1990; Seitz et al., 1991) and pseudogout (Miller and Brelsford, 1993); and to have a role in cystic fibrosis (McElvaney et al., 1992; Nakamura et al., 1992; Bedard et al., 1993). Therefore, modulation of IL-8 function appears to be good strategy to control a variety of pathological conditions.
Some progress has recently been made in identifying compounds capable of reducing IL-8 synthesis. Such compounds include IL-4, oxygen radical scavengers, secretory leukoprotease inhibitor and interferon gamma (Standiford et al., 1990; DeForge et al., 1992; McElvaney et al., 1992; Cassatella et al., 1993a; 1993b), however, such studies do not concern IL-8 inhibitors. Other diverse compositions, including protein kinase C inhibitors, IL-4, and anti-IL-8 antibodies, have also been reported to modulate IL-8 actions (Lam et al., 1990; Standiford et al., 1992; Mulligan et al., 1993). Unfortunately, these compounds are far from ideal as candidates for use as IL-8 inhibitors in a clinical setting.
Certain progress has also been made in identifying peptide IL-8 inhibitors, however, most of such work has focused on portions of the IL-8 molecule itself (Miller et al., 1990; Gayle et al., 1993). For example, the present inventors have shown that synthetic peptides, and particularly, IL-8 amino terminal peptides, inhibit IL-8 binding to neutrophils and neutrophil chemotaxis (Miller et al., 1990; Miller et al., 1993). An N-terminal pentapeptide IL-8 inhibitor has also been reported (Goodman et al., 1991). Unfortunately, to date, the inhibitory function of IL-8 derived peptides has proven incomplete and insufficient.
As particularly effective peptide inhibitors of CXC intercrines such as IL-8 have yet to be identified, it seems to be clear that compositions other than the IL-8 molecule itself now need to be investigated. The identification of peptide inhibitors capable of preferentially inhibiting neutrophil enzyme release in comparison to chemotaxis would be a particularly advantageous discovery as this would enable neutrophils to enter the lungs and defend against bacterial invasion and yet not cause tissue damage.